Glucocoticoids and cyclic AMP increase tyrsine aminotransferase (TAT) activity in rat liver following in vivo administration. The induction of enzyme activity by either inducer is the result of an increase in the level of functional tyrosine aminotransferase mRNA. The overall objective of this proposal is to elucidate the molecular mechanism(s) by which glucocorticoids and cyclic AMP regulate TAT catalytic and mRNA activity. These studies will utilize isolate rat liver parenchymal cell suspensions (hepatocytes) in which the induction of TAT activity is dependent upon the presence of both the glucocorticoid hormone and cyclic AMP. Intact and cytochalasin B-encleated cells will be used to discriminate between nuclear and cytoplasmic actions of the two inducers on TAT mRNA levels. The induction of TAT mRNA activity by protein synthesis inhibitors will also be investigated to determine the molecular basis and implications of this unusual observation. The interaction between glucocorticoids and cyclic AMP in regulating TAT mRNA levels will be examined from the point of view of a recently described steroid and cyclic AMP regulated phosphoprotein (SCARP) present in rat liver. These studies will investigate the status of SCARP under hormonal conditions leading to the induction of TAT mRNA activity to determine if SCARP is a direct link in the molecular sequence of events by which glucocorticoids and cyclic AMP exert their effect. The studies described above would be greatly facilitated if a complementary DNA (cDNA) hybridization probe for TAT mRNA was available. The preparation of TAT cDNA requires a pure mRNA template which will be prepared from rat liver polysomes by a combination of pyridoxamin phosphate affinity chromatography followed by immunochemical precipitation of TAT synthesizing ribosomes. The TAT cDNA transcribed from pure TAT mRNA will be utilized to quantitate TAT mRNA sequences by hybridization to total RNA prepared from liver cells treated under various hormonal conditions.